CN111607506B - Film type nucleic acid amplification micro-fluidic chip and preparation and application methods thereof - Google Patents
Film type nucleic acid amplification micro-fluidic chip and preparation and application methods thereof Download PDFInfo
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- CN111607506B CN111607506B CN202010528204.8A CN202010528204A CN111607506B CN 111607506 B CN111607506 B CN 111607506B CN 202010528204 A CN202010528204 A CN 202010528204A CN 111607506 B CN111607506 B CN 111607506B
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/14—Process control and prevention of errors
- B01L2200/141—Preventing contamination, tampering
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
- B01L2400/0478—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure pistons
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
- B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/06—Valves, specific forms thereof
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a film type nucleic acid amplification micro-fluidic chip and a preparation and application method thereof, relating to the technical field of biomolecule detection and comprising a plurality of reaction chambers connected through channels; the reaction chamber comprises a cracking chamber, a purification chamber, a pre-amplification chamber and an amplification chamber; the cracking cavity is provided with a sample inlet and is communicated with the purification cavity; the purification cavity is respectively communicated with a first liquid storage cavity preloaded with nucleic acid purification liquid and a second liquid storage cavity preloaded with nucleic acid eluent; the purification cavity is internally pre-packaged with magnetic beads coated with silicon dioxide and communicated with the pre-amplification cavity; the pre-amplification cavity is communicated with a third liquid storage cavity pre-filled with nucleic acid amplification diluent and is communicated with the amplification cavity; solves the problems that the cracking, purifying and amplifying processes in the prior art cannot be completely integrated on the same chip, the operation is complex and the pollution is easy to occur in the operation process.
Description
Technical Field
The invention relates to the technical field of biomolecule detection, in particular to a film type nucleic acid amplification micro-fluidic chip and a preparation and application method thereof.
Background
The nucleic acid-based diagnosis technology is one of the most powerful potential of the current molecular diagnosis technology, and has wide application in the fields of disease detection, food safety and the like. In the detection method of nucleic acid, polymerase Chain Reaction (PCR) technology is to amplify a specific nucleic acid fragment to thereby achieve the purpose of detecting a specific target nucleic acid fragment. In general, the detection of nucleic acids requires three steps: nucleic acid extraction, nucleic acid amplification, and nucleic acid detection.
The three steps of the traditional nucleic acid detection method are all discrete, and have the problems of long preparation and analysis time, easiness in pollution, sensitivity limitation, complex procedures and operation and the like, and the integration of nucleic acid extraction, amplification and detection by a microfluidic technology is helpful for solving the technical and analysis limitations related to nucleic acid detection in practical application.
The microfluidic chip adopted in the research at present is mostly only used for nucleic acid amplification, and can not integrate the cracking, purifying and amplifying processes on the same chip completely, and the microfluidic chip needs to be treated in advance in the operation process and then enters the chip through a sample inlet for nucleic acid amplification and detection, or needs to be manually added with reagents such as diluent in the operation process, so that the operation is complicated and the microfluidic chip is easy to pollute in the operation process.
Disclosure of Invention
The invention aims to provide a film type nucleic acid amplification micro-fluidic chip and a preparation and application method thereof, and provides an integrated film micro-fluidic chip which is used for solving the problems that cracking, purifying and amplifying processes cannot be integrated on the same chip completely, the operation is complicated and the pollution is easy in the operation process in the prior art.
In order to achieve the above object, the present invention provides a thin film type nucleic acid amplification microfluidic chip, comprising a plurality of reaction chambers connected by channels;
the reaction cavity comprises a cracking cavity, a purification cavity, a pre-amplification cavity and an amplification cavity;
the cracking cavity is provided with a sample inlet and is communicated with the purification cavity through a first channel;
the purification cavity is respectively communicated with a first liquid storage cavity preloaded with nucleic acid purification liquid and a second liquid storage cavity preloaded with nucleic acid eluent through a second channel and a third channel;
the purification cavity is internally pre-packaged with magnetic beads coated with silicon dioxide and is communicated with the pre-amplification cavity through a fourth channel;
the pre-amplification cavity is communicated with a third liquid storage cavity pre-filled with nucleic acid amplification diluent through a fifth channel and is communicated with the amplification cavity through a sixth channel.
Further, the second channel, the third channel and the fifth channel are respectively provided with a separation part for limiting the movement of the test sample;
and each channel is provided with a control valve for controlling the flow of the test sample.
Further, at least one redundant liquid cavity is communicated with the amplification cavity.
Further, the lysis chamber, the purification chamber, the pre-amplification chamber and the amplification chamber are vertically and sequentially arranged.
Further, the first liquid storage cavity is arranged on the first side of the purification cavity; the second liquid storage cavity is arranged on the second side of the purification cavity.
Further, a reaction plate with a plurality of through holes is arranged in the amplification cavity;
one side of the reaction plate is provided with a closed first film, the other side of the reaction plate is provided with a second film with small holes, and the through holes and the film layers on the two sides form a reaction tank;
the small holes are in one-to-one correspondence with the through holes;
the reaction tanks are provided with at least two groups, and each group at least comprises three groups.
Further, the chip is formed by sealing at least two layers of films.
Further, the film is made of a transparent flexible polymer material, and the polymer material is one of polycarbonate, polypropylene, polyethylene terephthalate or thermoplastic elastomer material.
Further, the isolation part is one or more of a physical isolation belt, a one-way film or a one-way valve.
Further, two redundant liquid cavities are arranged and correspondingly arranged at two sides of the amplification cavity.
Further, the first and fourth channels are connected to the top and bottom sides of the purification chamber, respectively.
In order to achieve the above purpose, the invention also provides a preparation method of the film type nucleic acid amplification micro-fluidic chip, which is used for preparing any one of the film type nucleic acid amplification micro-fluidic chip, and is prepared by sealing two or more layers of films, wherein the sealing mode adopts one or more modes of laser welding, hot press sealing, high-strength chemical glue bonding or ultrasonic welding.
In order to achieve the above object, the present invention also provides an application method of a thin film type nucleic acid amplification micro-fluidic chip, using any one of the above thin film type nucleic acid amplification micro-fluidic chips, comprising the steps of:
step one: the method comprises the steps of enabling a test sample to enter a chip through a sample inlet and enter a cracking cavity, cracking the test sample, and enabling the cracked test sample to enter a purification cavity;
step two: controlling nucleic acid purification liquid pre-buried in the first liquid storage cavity to squeeze into the purification cavity to be mixed with the cracked test sample, and releasing nucleic acid molecules in the test sample;
step three: nucleic acid eluent pre-loaded in the second liquid storage cavity enters the purification cavity for nucleic acid extraction to obtain liquid with nucleic acid molecules;
step four: extruding the liquid with nucleic acid molecules into a pre-amplification cavity, and pre-amplifying under a preset temperature environment;
step five: introducing nucleic acid diluent pre-stored in the third liquid storage cavity into a nucleic acid pre-amplification cavity to obtain a mixed solution, and controlling the mixed solution to enter an amplification cavity to perform nucleic acid amplification reaction.
In the second step, the purification cavity and the first liquid storage cavity are repeatedly extruded to enable the mixed liquid to move back and forth in the purification cavity and the first liquid storage cavity so as to mix the nucleic acid purification liquid, the test sample and the magnetic beads, and then nucleic acid molecules in the test sample are released.
Furthermore, in the step four, the nucleic acid is extracted, and the liquid is controlled to move back and forth by extruding the purifying cavity and the second liquid storage cavity, so that the nucleic acid eluent dissolves the nucleic acid molecules adsorbed on the surfaces of the magnetic beads.
Further, after the mixed liquid is extruded into the amplification cavity, the internal reference targets or the detection targets are buried in each reaction cavity in advance, and after the mixed liquid fills each reaction tank, the redundant liquid is controlled to enter the redundant liquid cavity.
Furthermore, after the nucleic acid amplification is completed in the step five, the specific target is qualitatively or quantitatively judged by adopting a fluorescence light source to excite and according to the fluorescence intensity in each reaction tank.
The invention provides a film type nucleic acid amplification micro-fluidic chip and a preparation method and an application method thereof, wherein a chip made of a film is provided with a cracking cavity, a purifying cavity, a pre-amplification cavity and an amplification cavity from top to bottom in sequence; in the use process of the chip, after a sample enters the cracking cavity, a closed space is formed inside the chip, and cracking, purifying and amplifying reactions are sequentially carried out, so that the problems that the cracking, purifying and amplifying processes cannot be completely integrated on the same chip in the prior art, the operation is complex and the sample is easy to pollute in the operation process are solved, the sample inlet and outlet can be realized, the whole detection process only needs to be carried out once, and the interference of human factors is reduced.
Drawings
FIG. 1 is a schematic diagram of a thin film nucleic acid amplification microfluidic chip according to an embodiment of the present invention;
FIG. 2 is a schematic structural diagram of a reaction plate in a first embodiment of a membrane-type nucleic acid amplification microfluidic chip according to the present invention;
FIG. 3 is a flow chart of an embodiment of a method for applying a thin film nucleic acid amplification microfluidic chip according to the present invention;
fig. 4 is a block diagram showing the action of a pneumatic piston in a third example of an application method of a thin film type nucleic acid amplification microfluidic chip according to the present invention.
Reference numerals:
1. lysis chamber 2, purification chamber 3, pre-amplification chamber 4, amplification chamber
5. A first liquid storage cavity 6, a second liquid storage cavity 7, a third liquid storage cavity 8 and a redundant liquid cavity
9. Control valve 10, spacer 12, closure cap 42, and apertured film layer
43. Reaction plate 44, sealing film layer
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It will be understood that, although the terms "first," "second," etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another element. Thus, a "first" element may be termed a "second" element without departing from the teachings of the present embodiment.
It should be noted that the words "front", "back", "left", "right", "upper" and "lower" used in the following description refer to directions in the drawings, and the words "bottom" and "top", "inner" and "outer" refer to directions toward or away from, respectively, the geometric center of a particular component.
It is also noted that in the embodiments described below, a sample means a solution of one or more molecules of a cell, cell lysate or cell extract, cellular material or viral material (e.g., a polypeptide or nucleic acid); or contain non-naturally occurring nucleic acids (e.g., cDNA), but may be any external solution that may or may not contain pathogen cells, cellular components, or nucleic acids.
Embodiment one:
in a specific embodiment, a thin film type nucleic acid amplification micro-fluidic chip is provided, referring to fig. 1, the chip is formed by sealing at least two layers of thin films, and comprises a plurality of reaction chambers connected by channels, in the specific embodiment, two layers or three layers of plastic thin films are adopted to manufacture the chip, and the thin films are adopted to manufacture the chip, which is different from a Polydimethylsiloxane (PDMS) chip adopted in the prior study and a plastic chip adopted in the actual product, so that the flow direction of a sample in the chip is easier to control, and the whole process of nucleic acid extraction and nucleic acid amplification is realized.
In a preferred embodiment, the membrane is a transparent flexible polymeric material, which is one of polycarbonate, polypropylene, polyethylene terephthalate or thermoplastic elastomer material, and it is particularly noted that it is a transparent flexible material, which is primarily convenient for a technician to intuitively track the reaction state of the sample during operation, and flexibility is primarily convenient for the control system to move the sample and reagent mixture between the reaction chambers by applying pressure, illustratively pneumatic pressure, to the reaction chambers and channels during use in conjunction with the control system, and accordingly, in embodiments employing pressure, the "flexibility" is defined in this embodiment as the use of pressure to squeeze the reaction chambers and channels without cracking, splitting, etc.
In this embodiment, the reaction chamber includes a lysis chamber, a purification chamber, a pre-amplification chamber, and an amplification chamber sequentially disposed from top to bottom; each reaction chamber can be set to any shape, preferably spherical to reduce the damage risk that leads to under the exogenic action in the follow-up use, schizolysis chamber the purification chamber the preamplification chamber reaches the vertical setting gradually in amplification chamber can improve space utilization, and simultaneously supplementary action of gravity accelerates liquid flow rate in the reaction, be equipped with on the schizolysis chamber and have the inlet of closing cap, and with communicate through first passageway on the purification chamber, in this chip use, make the inside confined space that forms of chip after the sample gets into the schizolysis chamber, carry out schizolysis, purification, amplification reaction in proper order.
The purification cavity is respectively communicated with a first liquid storage cavity preloaded with nucleic acid purification liquid and a second liquid storage cavity preloaded with nucleic acid eluent through a second channel and a third channel; the first liquid storage cavity is arranged on the first side of the purification cavity; the second liquid storage cavity is arranged on the second side of the purification cavity, namely the first liquid storage cavity and the second liquid storage cavity are oppositely arranged on two sides of the purification cavity, so that the nucleic acid purification liquid and the nucleic acid eluent can be controlled to enter the purification cavity in the follow-up operation, and the interference between the two processes is reduced; the purification cavity is internally pre-packaged with magnetic beads coated with silicon dioxide and communicated with the amplification cavity through a fourth channel, the magnetic beads coated with silicon dioxide are used for adsorbing nucleic acid molecules, in the subsequent operation process, nucleic acid purification liquid and a test sample are mixed to obtain magnetic beads adsorbed with nucleic acid molecules, and then nucleic acid eluent is used for cleaning the magnetic beads to realize extraction of nucleic acid; the third liquid storage cavity which is communicated with the amplification cavity through a fifth channel and is preloaded with nucleic acid amplification diluent is communicated with the amplification cavity through a sixth channel.
In this embodiment, the amplification cavity is further communicated with at least one redundant liquid cavity, and the redundant liquid cavity is mainly used for collecting redundant liquid in the amplification cavity, so that a great amount of liquid is retained in the amplification cavity to affect the nucleic acid amplification reaction process.
It should be noted that, the second channel, the third channel and the fifth channel are all provided with a separation part for limiting the movement of the test sample, and the separation part is mainly used for preventing the movement of the pre-loaded nucleic acid purification solution, the nucleic acid eluent and the nucleic acid amplification diluent, and controlling each liquid to participate in the reaction at each stage of the reaction. In this embodiment, in order to mix two volumes of liquid in different reaction chambers, the control valves of the sealing connection channels are activated and the reaction chambers are alternately pressurized to force liquid back and forth through the channels into the respective reaction chambers to mix the liquid therein, and the control valves may have various shapes and sizes and may be various physical valves such as butterfly valves and ball valves. The chip in this embodiment is typically used in conjunction with a control device, and the various partitions, control valves, and reaction chambers may be pressurized by a pneumatic control device, although it should be understood that other means of providing pressure to the reaction chambers may be used, including various electromechanical actuators, etc.
The physical isolation part is formed by the hot-pressing belt, the unidirectional isolation film or the unidirectional valve, so that the reagent can be pre-buried in the film chip, the isolation part can be extruded and broken by means of the pneumatic control piston when the reaction is needed, and the two physically isolated areas are communicated, so that the reagent in the film chip is pre-buried.
In a preferred embodiment, referring to FIG. 3, a reaction plate with a plurality of through holes is disposed in the amplification chamber; one side of the reaction plate is a closed film layer, the other side of the reaction plate is a film layer with small holes, and the through holes and the film layers on the two sides form a reaction tank; the small holes are in one-to-one correspondence with the through holes; the reaction tanks are arranged in at least two groups, each group comprises at least three, in the specific embodiment, the reaction tanks are arranged in five groups, each group comprises three, two adjacent groups of reaction tanks are arranged in parallel relatively, each group is internally embedded with a preset target and enzymes and primers required by amplification reaction, any group is internally embedded with an internal reference target, other groups of reaction tanks can be embedded with the same test target, and different test targets can be used for realizing detection of various germs.
In the embodiment, the small holes are used for enabling the liquid with the nucleic acid of the test sample to enter the reaction tank, limiting the liquid entering the reaction tank to flow out to a certain extent, preventing the interaction of the liquid among different through holes, further ensuring the smooth progress of the amplification reaction, arranging three reaction tanks into a group and embedding the same test targets, and reducing the occurrence of inaccurate subsequent test results caused by the influence of external factors on the reaction process.
In a preferred embodiment, the first channel and the fourth channel are respectively connected to the top side and the bottom side of the purification chamber, i.e. as shown in the figure, the first channel and the fourth channel are both in an inverted "L" shape, and in the above embodiment, the arrangement is mainly used for preventing the liquid from flowing in the channels between the lysis chamber, the purification chamber, the pre-amplification chamber and the amplification chamber directly without applying external force, giving a certain restriction to the liquid flow, so as to be beneficial to controlling the flow direction of the liquid in the chip, and simultaneously, when the pressure of each reaction chamber is given to enable the liquid to flow, the liquid flow speed is faster due to the action of gravity, thereby further saving the reaction time and improving the working efficiency.
The chip in the embodiment integrates the functions of nucleic acid extraction, purification and amplification on the same film chip, and can realize integrated nucleic acid detection, the integrated film chip mainly comprises four functional areas of cracking, purification, pre-amplification and detection, and the four functional areas are sequentially arranged from top to bottom.
Embodiment two:
the application provides a preparation method of a film type nucleic acid amplification micro-fluidic chip, which is used for preparing the film type nucleic acid amplification micro-fluidic chip in the first embodiment, specifically, the film type nucleic acid amplification micro-fluidic chip is prepared by sealing two or more layers of films, and the sealing mode adopts one or more modes of laser welding, hot press sealing, high-strength chemical glue bonding or ultrasonic welding.
The film can be cut before sealing, so that the thickness of the film type nucleic acid micro-fluidic chip can be controlled more reasonably, and the temperature control efficiency can be improved; because the flexibility of the film is better, the chip edge is not easy to warp or break after being processed, the sealing performance is good, the production and processing can be carried out by adopting various modes according to the equipment condition of actual production, for example, the sealing is carried out by adopting a welding mode, the sealing efficiency can be improved, compared with other integrated detection chip structures in the prior art, the structure processing technology designed in the way is simpler, and the processing cost can be greatly reduced.
Embodiment III:
the application provides an application method of various film type nucleic acid amplification micro-fluidic chips, referring to fig. 2, one of the film type nucleic acid amplification micro-fluidic chips according to the embodiment is used, and the film type nucleic acid amplification micro-fluidic chip can be matched with a control system for use in a detection process, and comprises the following steps:
step one: the method comprises the steps of enabling a test sample to enter a chip through a sample inlet and enter a cracking cavity, cracking the test sample, and enabling the cracked test sample to enter a purification cavity;
specifically, the chip is placed in a control system, and the control piston or partition closes the corresponding fluid passage. The integrated thin film chip can be injected with a sample from a sample inlet through a matched injector, the whole chip is sealed by a sealing cap after the sample is injected, a sealing space for reaction is formed in the chip, and the test sample enters the cracking cavity. The process of cracking the test sample in the above steps can be realized by controlling the temperature or extruding the cracking cavity.
Step two: opening an isolation region on the second channel, extruding nucleic acid purification liquid pre-buried in the first liquid storage cavity into the purification cavity, fully mixing the nucleic acid purification liquid after the nucleic acid purification liquid enters the purification cavity, releasing nucleic acid molecules in the test sample, extruding mixed liquid into the first liquid storage cavity, and closing the isolation region on the second channel;
as a further improvement of the invention, in the third step, the mixed liquid is repeatedly extruded in the purification cavity and the first liquid storage cavity to make the mixed liquid move back and forth in the purification cavity and the first liquid storage cavity so as to fully mix the nucleic acid purification liquid, the test sample and the magnetic beads, and the nucleic acid molecules in the test sample are released to be adsorbed on the magnetic beads.
In the above embodiment, the movement of the reaction liquid is realized by extruding the film, so that the full mixing of the liquid and the magnetic beads is realized, the magnetic beads can fully adsorb the nucleic acid molecules, and further, the full release of the nucleic acid molecules is realized, and the reciprocating movement mode is different from the mode of arranging a plurality of purification cavities in the prior art, so that the use space of a chip is saved, and the smooth realization of the purification process is ensured.
Step three: opening an isolation region on the third channel, extruding the nucleic acid eluent pre-loaded in the second liquid storage cavity into a purification cavity, dissolving nucleic acid molecules adsorbed on the surfaces of the magnetic beads, and extracting nucleic acid to obtain liquid with the nucleic acid molecules;
as a further improvement of the invention, the nucleic acid molecules adsorbed on the surface of the magnetic beads are dissolved in the third step, and the mixed liquid is moved back and forth by extruding the purification cavity and the second liquid storage cavity.
In the above embodiment, the second step is to adsorb the nucleic acid molecules on the surface of the magnetic beads, and the third step is to dissolve the adsorbed nucleic acid molecules to obtain a solution containing the nucleic acid molecules so as to perform subsequent amplification reaction, and the method of back and forth mixing similar to that in the second step is simple and convenient to operate, and the efficiency is improved.
Step four: opening a control valve on the fourth channel, extruding liquid with nucleic acid molecules into a pre-amplification cavity to perform pre-amplification under a preset temperature environment, and completing the pre-amplification;
in the above embodiment, the pre-amplification is implemented by thermal cycling under a preset temperature environment.
Step five: opening an isolation region on the fifth channel, introducing nucleic acid diluent pre-stored in the third liquid storage cavity into the nucleic acid pre-amplification cavity, mixing, opening a control valve on the sixth channel, and extruding the mixed liquid into the amplification cavity to perform nucleic acid amplification reaction.
The primer for PCR reaction, DNTP (deoxyribonucleoside triphosphate) and enzyme required for the reaction are pre-buried in each reaction tank in the five amplification chambers in the steps so as to meet the reaction conditions for completing the nucleic acid amplification in the reaction tanks.
In the above embodiment, after the mixed liquid is extruded into the amplification cavity, the internal reference targets or the detection targets are buried in each reaction cavity in advance, and after the mixed liquid fills each reaction cavity, the amplification cavity is extruded to make the redundant liquid enter the redundant liquid cavity.
And step five, exciting the nucleic acid by adopting a fluorescent light source after completing nucleic acid amplification, and qualitatively or quantitatively judging a specific target according to the fluorescent intensity in each reaction tank.
In the above embodiment, the quantitative determination of the nucleic acid detection result can be realized by using a fluorescent quantitative PCR technique, wherein the real-time fluorescent quantitative PCR (real-time quantitative PCR) technique is to heat a specific fluorescent dye or probe in a PCR reaction system, the change of a fluorescent signal truly reflects the increase of a template in the system, and the quantitative purpose is achieved by detecting the fluorescent signal; each target in the amplification cavity is provided with 3 reaction cavities, and the same target detection reaction can be simultaneously repeated for 3 times, in addition, an internal reference target is simultaneously set in the amplification cavity, the amplified fluorescence intensities in three reaction tanks corresponding to the internal reference target are judged, and the detection process can be determined to be effective only after more than two fluorescence intensities of three positions of the internal reference target reach the set threshold. For each specific detection target, the presence of the target in the sample can be determined only after two or more fluorescence intensities of three reaction cells of the target reach a set threshold. If only one of the fluorescence intensities of the three reaction chambers reaches the set threshold, the sample is judged to be suspected, and the arrangement can provide guarantee for the accuracy of the detection result.
The quantitative standard may have different forward or reverse primers or the same forward and reverse primers and by way of example and not limitation, fluorescent excitation in this application includes, for example, chemiluminescence, bioluminescence, radioluminescence, electroluminescence, electrochemiluminescence, mechanoluminescence, crystallization luminescence, thermoluminescence, sonoluminescence, and other forms of photoluminescence, enzymatic, radioactivity, and the like, with specific fluorescent probes or other identifiable labels.
The chip is matched with a control system, and liquid in and out or movement in each reaction cavity can be controlled by a pneumatic piston extrusion mode.
Taking a specific example of detecting germs of respiratory tract infection, referring to fig. 1 and fig. 4, the detection process is as follows:
respectively embedding a lysis solution, a nucleic acid purification solution, a nucleic acid eluent and an amplification diluent in the lysis cavity, the first liquid storage cavity, the second liquid storage cavity and the third liquid storage cavity; embedding yeast or phage in a first group of reaction tanks in the amplification cavity as an internal reference target, and embedding pertussis bacillus, chlamydia pneumoniae or mycoplasma pneumoniae and the like in other groups of reaction tanks as detection targets; the purification chamber is internally provided with a plurality of magnetic beads, and each reaction chamber is also assumed to be provided with a pneumatic control piston for controlling extrusion or extrusion.
Injecting a sample into the integrated film chip through a matched injector, sealing the whole chip by using a sealing cap after the sample is injected, putting the chip into a control system, when the sample enters a cracking cavity 1, controlling a control valve between the cracking cavity 1 and a purifying cavity 2 to be closed, reacting in the cracking cavity to realize cracking, opening the control valve after the cracking is completed, starting a pneumatic piston 11 to extrude the cracked sample into the purifying cavity 2, opening a control pneumatic piston 51 to extrude a nucleic acid purifying liquid pre-embedded in a first liquid storage cavity 5 into the purifying cavity 2, opening a separation part in the extrusion process, closing the control pneumatic piston 51 after the nucleic acid purifying liquid enters the purifying cavity 2 and is mixed with magnetic beads coated by silicon dioxide, opening the pneumatic piston 21, extruding the sample into the first liquid storage cavity 51 again, by controlling the starting and closing of the pneumatic piston 21 and the pneumatic piston 51, the cracked mixed solution of the sample and the magnetic beads is back extruded and mixed in the first liquid storage cavity 5 and the purifying cavity 2, after the sample and the magnetic beads coated with silicon dioxide are fully mixed, an electromagnet at the purifying cavity 2 is opened, the magnetic beads stay in the purifying cavity 2, the liquid is extruded into the first liquid storage cavity 5, a control valve between the first liquid storage cavity 5 and the purifying cavity 2 is closed, an isolation part between the second liquid storage cavity 6 and the purifying cavity 2 is opened, the nucleic acid eluent pre-loaded in the second liquid storage cavity 6 is extruded into the purifying cavity 2 through the pneumatic piston 61, the magnetic beads and the nucleic acid eluent are fully mixed through the pneumatic piston 21 and the pneumatic piston 61, after the nucleic acid molecules adsorbed on the surface of the magnetic beads are fully dissolved, the liquid is reserved in the purifying cavity 2, the control valve between the second liquid storage cavity 6 and the purifying cavity 2 is closed, opening a control valve between the pneumatic piston 21 and the purification cavity 2 and the pre-amplification cavity 3, extruding liquid into the pre-amplification cavity 3, closing the control valve after the liquid enters the pre-amplification cavity 3, carrying out thermal cycle pre-amplification on the liquid with the test sample in the pre-amplification cavity 3, opening an isolation part between the pre-amplification cavity 3 and the third liquid storage cavity 7 after the pre-amplification is finished, introducing nucleic acid diluent into the nucleic acid pre-amplification cavity 3 through the pneumatic piston 71 on the third liquid storage cavity 7, fully mixing the diluent and a pre-amplification product by shaking back and forth through the pneumatic piston 71 and the pneumatic piston 31, opening the control valve between the pre-amplification cavity 3 and the amplification cavity 4 after the mixing is finished, introducing mixed liquid into the amplification cavity 4, opening the pneumatic piston 41 on the amplification cavity 4 after each reaction tank is filled with the solution, and extruding redundant liquid into the redundant liquid cavity 8. And (3) performing thermal cycle amplification in the reaction tank, after the amplification is finished, exciting by a fluorescent light source, recording the fluorescent intensity in each hole in the amplification cavity 4 by a camera or a CCD (charge coupled device image sensor), and determining whether a specific detection target exists in the sample according to the target type set at a specific position.
It should be noted that, the pneumatic piston 11 is correspondingly arranged on the cracking 1, and the pneumatic piston 21 is correspondingly arranged on the purifying cavity 2; the pre-amplification cavity 3 is correspondingly provided with a pneumatic piston 31; pneumatic pistons 41 are correspondingly arranged on the amplification chambers 4; the first liquid storage cavity 5, the second liquid storage cavity 6 and the third liquid storage cavity are correspondingly provided with pneumatic pistons 51, 61 and 71; the pneumatic pistons correspondingly arranged on the reaction chambers can be opened to give pressure to the reaction chambers, and the pressure is removed when the pneumatic pistons are closed.
The foregoing embodiment numbers of the present invention are merely for the purpose of description, and do not represent the advantages or disadvantages of the embodiments.
From the above description of the embodiments, it will be clear to those skilled in the art that the above-described embodiment method may be implemented by means of software plus a necessary general hardware platform, but of course may also be implemented by means of hardware, but in many cases the former is a preferred embodiment.
The foregoing description is only of the preferred embodiments of the present invention, and is not intended to limit the scope of the invention, but rather is intended to cover any equivalents of the structures or equivalent processes disclosed herein or in the alternative, which may be employed directly or indirectly in other related arts.
Claims (16)
1. A film type nucleic acid amplification micro-fluidic chip is characterized in that:
comprises a plurality of reaction chambers connected by channels;
the reaction cavity comprises a cracking cavity, a purification cavity, a pre-amplification cavity and an amplification cavity;
the cracking cavity is provided with a sample inlet and is communicated with the purification cavity through a first channel;
the purification cavity is respectively communicated with a first liquid storage cavity preloaded with nucleic acid purification liquid and a second liquid storage cavity preloaded with nucleic acid eluent through a second channel and a third channel;
the purification cavity is internally pre-packaged with magnetic beads coated with silicon dioxide and is communicated with the pre-amplification cavity through a fourth channel;
the pre-amplification cavity is communicated with a third liquid storage cavity pre-filled with nucleic acid amplification diluent through a fifth channel and is communicated with the amplification cavity through a sixth channel;
the lysis cavity, the purification cavity, the pre-amplification cavity and the amplification cavity are vertically and sequentially arranged;
a reaction plate with a plurality of through holes is arranged in the amplification cavity;
one side of the reaction plate is provided with a closed first film, the other side of the reaction plate is provided with a second film with small holes, and the through holes and the film layers on the two sides form a reaction tank;
the small holes are in one-to-one correspondence with the through holes.
2. The thin film nucleic acid amplification microfluidic chip of claim 1, wherein:
the second channel, the third channel and the fifth channel are respectively provided with an isolation part for limiting the movement of the test sample;
and each channel is provided with a control valve for controlling the flow of the test sample.
3. The thin film nucleic acid amplification microfluidic chip of claim 1, wherein:
the amplification cavity is also communicated with at least one redundant liquid cavity.
4. The thin film nucleic acid amplification microfluidic chip of claim 1, wherein:
the first liquid storage cavity is arranged on the first side of the purification cavity; the second liquid storage cavity is arranged on the second side of the purification cavity.
5. The thin film nucleic acid amplification microfluidic chip of claim 1, wherein:
the reaction tanks are provided with at least two groups, and each group at least comprises three groups.
6. The thin film nucleic acid amplification microfluidic chip of claim 1, wherein:
the chip is formed by sealing at least two layers of films.
7. The thin film nucleic acid amplification microfluidic chip of claim 6, wherein:
the film is made of transparent flexible polymer material, and the polymer material is one of polycarbonate, polypropylene, polyethylene terephthalate or thermoplastic elastomer material.
8. The thin film nucleic acid amplification microfluidic chip of claim 2, wherein:
the isolation part is one or more of a physical isolation belt, a one-way film or a one-way valve.
9. A thin film nucleic acid amplification microfluidic chip as defined in claim 3, wherein:
the redundant liquid chambers are arranged in two and correspondingly arranged on two sides of the amplification chamber.
10. The thin film nucleic acid amplification microfluidic chip of claim 1, wherein:
the first and fourth channels are connected to the top and bottom sides of the purification chamber, respectively.
11. A method for preparing a thin film type nucleic acid amplification micro-fluidic chip, which is used for preparing the thin film type nucleic acid amplification micro-fluidic chip as set forth in any one of claims 1 to 10, and is characterized in that:
the sealing is made by adopting two or more layers of films, and the sealing mode adopts one or more modes of laser welding, hot-press sealing, high-strength chemical glue bonding or ultrasonic welding.
12. An application method of a thin film type nucleic acid amplification micro-fluidic chip, which uses any one of the thin film type nucleic acid amplification micro-fluidic chips as described in claims 1-10, and is characterized by comprising the following steps:
step one: the method comprises the steps of enabling a test sample to enter a chip through a sample inlet and enter a cracking cavity, cracking the test sample, and enabling the cracked test sample to enter a purification cavity;
step two: controlling nucleic acid purification liquid pre-buried in the first liquid storage cavity to squeeze into the purification cavity to be mixed with the cracked test sample, and releasing nucleic acid molecules in the test sample;
step three: nucleic acid eluent pre-loaded in the second liquid storage cavity enters the purification cavity for nucleic acid extraction to obtain liquid with nucleic acid molecules;
step four: extruding the liquid with nucleic acid molecules into a pre-amplification cavity, and pre-amplifying under a preset temperature environment;
step five: introducing nucleic acid diluent pre-stored in the third liquid storage cavity into a nucleic acid pre-amplification cavity to obtain a mixed solution, and controlling the mixed solution to enter an amplification cavity to perform nucleic acid amplification reaction.
13. The method as recited in claim 12, wherein:
in the second step, the nucleic acid purification liquid, the test sample and the magnetic beads are mixed by repeatedly squeezing the purification cavity and the first liquid storage cavity to enable the mixed liquid to move back and forth in the purification cavity and the first liquid storage cavity, so that nucleic acid molecules in the test sample are released.
14. The method as recited in claim 12, wherein:
and in the fourth step, the extracted nucleic acid is controlled by the extrusion purification cavity and the second liquid storage cavity to make the liquid move back and forth so that the nucleic acid eluent dissolves the nucleic acid molecules adsorbed on the surfaces of the magnetic beads.
15. The method as recited in claim 12, wherein:
and step five, extruding the mixed liquid into an amplification cavity, burying an internal reference target or a detection target in each reaction cavity in advance, and controlling the redundant liquid to enter the redundant liquid cavity after the mixed liquid fills each reaction tank.
16. The method as recited in claim 12, wherein:
and step five, exciting the nucleic acid by adopting a fluorescent light source after completing nucleic acid amplification, and qualitatively or quantitatively judging a specific target according to the fluorescent intensity in each reaction tank.
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| CN113244970B (en) * | 2021-01-07 | 2022-07-19 | 中国检验检疫科学研究院 | Microfluidic chip for nucleic acid extraction and PCR detection and application thereof |
| CN113174324B (en) * | 2021-01-15 | 2022-03-11 | 北京中科生仪科技有限公司 | PCR reaction device |
| CN113174308B (en) * | 2021-01-15 | 2021-12-14 | 北京中科生仪科技有限公司 | Purification amplification device |
| CN114768894B (en) * | 2021-01-22 | 2023-08-11 | 中国科学院上海微***与信息技术研究所 | Detection chip and detection method |
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| CN113736643A (en) * | 2021-09-23 | 2021-12-03 | 吉特吉生物技术(苏州)有限公司 | Micro-fluidic chip for nucleic acid amplification detection |
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| CN114405449A (en) * | 2022-02-19 | 2022-04-29 | 鲍熙来 | Chemical experiment device based on micro-fluidic |
| CN116891800A (en) * | 2022-03-31 | 2023-10-17 | 恒泰医疗有限公司 | Chip device and instrument for detecting nucleic acid and application thereof |
| CN114733587B (en) * | 2022-04-01 | 2024-02-20 | 合肥诺迈基生物科技有限公司 | Fluorescence detection chip, fluorescence detection system, fluorescence detection method and application thereof |
| CN115287176A (en) * | 2022-09-30 | 2022-11-04 | 潍坊安普未来生物科技有限公司 | Two-step isothermal nucleic acid amplification detection microfluidic device |
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